Methods for judging the possibility of the onset of bovine leukemia and the resistance thereto

ABSTRACT

A method for judging a possibility of the onset of bovine leukemia caused by bovine leukemia virus BLV, wherein a bovine individual, in which an amino acid sequence defined by the amino acid numbers 75 to 78 of the beta1 domain of the bovine MHC Class II DRbeta chain is Val-Asp-Thr-Tyr, is judged to have a possibility of the onset of the leukemia; and a method for judging a resistance to the onset of bovine leukemia caused by the bovine leukemia virus BLV, wherein a bovine individual, in which an amino acid defined by the amino acid number 78 of the beta1 domain of the bovine MHC Class II DRbeta chain is Val, is judged to have a resistance to the onset of the leukemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/147,550,[now U.S. Pat. No. 6,090,540], Apr. 23, 1999, which is the NationalStage of International Application No. PCT/JP97/02485, filed Jul. 17,1997, and claims priority of Japanese Application No. 8-190933, filedJul. 19, 1996 and Japanese Application No. 9-77979, filed Mar. 28, 1997.The entire disclosure of Application Ser. No. 09/147,550 is consideredas being part of the disclosure of this application, and the entiredisclosure of Application Ser. No. 09/147,550 is expressly incorporatedby reference herein in its entirety.

TECHNICAL FIELD

The present invention relates to a method for judging a possibility ofthe onset of bovine leukemia caused by bovine leukemia virus BLV and aresistance to the onset of the leukemia.

BACKGROUND ART

The major histocompatibility antigens (MHC antigens) are moleculesinvolved in self-nonself differentiation in the defense mechanism of theliving body against infection. They are classified into Class I moleculecomposed of α chain and β2M, and class II molecule composed of α chainand β chain. A groove for trapping an antigen peptide is present on theα1 and α2 domains, and also on the α1 and β1 domains. They are featuredto have the T cell receptor recognize only a fragmented peptide trappedin the groove, thereby achieve cell death (cellular immunity) by CD8+cells which have recognized the class I antigens, as well as inducemainly antibody production (humoral immunity) by CD4+ cells which haverecognized the class II antigens.

The MHC genes constitute a gene group most full of polymorphism, and thelocations of pockets, shapes, sizes and properties of the peptidetrapping grooves are different among haplotypes. It is considered thatassociation conditions of the trapped fragment peptides may varydepending on these differences, which decide immune response and diseasesensitivity of each individual. The correlation between the MHChaplotypes and a resistance to a disease (disease insusceptibility) or apossibility of the onset of a disease (disease susceptibility) has beenreported, for example, as to human immune deficiency virus (HIV), humanT cell leukemia virus (HTLV) and malaria.

As for the bovine MHC (BoLA) class II genes, existence of DQA, DQB, DRA,DRB, DNA, DOB, DYA, and DYB genes has been estimated. DRB3, inter alia,which is one of the three genes (DRB1 to B3) identified on the DRBgenetic locus, has been known to encode a functional protein, andexistence of 73 alleles has been revealed so far. However, there isalmost no report about correlation between bovine infectious diseasesand the bovine MHC (BoLA) haplotypes.

In particular, as to the bovine leukemia virus (BLV), which has the genePX that regulates virus proliferation in the same manner as the humanimmunodeficiency virus (HIV) and is a retrovirus most related to HTLV-I,a research group in the United States has reported its relationship withthe bovine MHC (BoLA) haplotypes mainly focusing on disease resistance;however, its relationship with possibility of onset of leukemia has notbeen reported. The ratio of cattle infected by this virus (infectionrate in Japan) is 10-20%, and 1-2% of the infected cattle developextremely malignant endemic bovine leukemia and die after a long latentperiod of 10-15 years. Therefore, economic loss of stockbreeders causedby the virus is very serious. If a possibility of the onset of leukemiain cattle after BLV infection can be evaluated by the analysis of bovineMHC (BoLA) haplotypes, it becomes possible to preliminarily selectdisease resistant cattle for breeding, and it is expected that extremelysafe cattle breeding can be continued.

Accordingly, an object of the present invention is to elucidate therelationship between the bovine leukemia virus (BLV) and the bovine MHC(BoLA) haplotypes, and to provide a method for convenient judgement of apossibility of the onset of leukemia of a cattle caused by the bovineleukemia virus (BLV) and a resistance to the onset of the leukemia bymeans of genetic engineering techniques. Another object of the presentinvention is to provide a primer set useful for the aforementionedmethod for judgement.

DISCLOSURE OF THE INVENTION

The inventors of the present invention previously analyzed the structureof DRB gene locus among the bovine MHC (BoLA) class II genes, andreported the structures of DRB3 gene (BoLA-DRB3) and the gene productthereof (Biochem. Biophys. Res. Commun., 209, pp.981-988, 1995). Theinventors further studied the function of the gene and found that aportion is present, whose amino acid sequence is distinctly differentbetween a cattle developing the leukemia and a cattle not developing thedisease, in the gene product from the second exon (β1 domain) ofBoLA-DRB3 showing particularly noticeable polymorphism. They also foundthat the amino acid substitutions directly correlated with diseasesusceptibility to BLV and disease resistance The present invention wasachieved on the basis of these findings.

The present invention thus provides a method for judging a possibilityof the onset of bovine leukemia caused by bovine leukemia virus BLV,wherein a bovine individual, in which an amino acid sequence defined bythe amino acid number from 75 to 78 of the β0 1 domain of the bovine MHCClass II DRβ chain is Val-Asp-Thr-Tyr, (SEQ ID NO:9) is judged to have apossibility of the onset of the leukemia. As preferred embodiments ofthe method of the present invention, there are provided theaforementioned method which is applied to a cattle infected by thebovine leukemia virus BLV; and the aforementioned method wherein abovine individual, in which an amino acid sequence defined by the aminoacid numbers 75-78 of the β1 domain of the bovine MHC Class II DRβ chainis Val-Asp-Thr-Tyr (SEQ ID NO:9) in both of the alleles, is judged tohave a risk of the onset.

According to another embodiment of the method of the present invention,there is provided a method for judging a possibility of the onset ofbovine leukemia caused by the bovine leukemia virus BLV, which comprisesthe steps of:

(1) amplifying genomic DNA isolated from a bovine individual by thepolymerase chain reaction (PCR) to prepare a PCR product containing aDNA coding for a part or full length of the β1 domain of the bovine MHCClass II DRβ chain, and

(2) judging that the bovine individual, in which an amino acid sequencecorresponding to the amino acid number from 75 to 78 of the β1 domain ofthe bovine MHC Class II DRβ chain is Val-Asp-Thr-Tyr (SEQ ID NO:9) inthe amino acid sequence encoded by the DNA contained in the PCR product,has a possibility of the onset of the leukemia. A preferred embodimentof the aforementioned method comprises a step of digesting the PCRproduct by using PstI.

According to another aspect of the present invention, there is provideda method for judging a resistance to the onset of bovine leukemia causedby the bovine leukemia virus BLV, wherein a bovine individual, in whichan amino acid defined by the amino acid number 78 of the β1 domain ofthe bovine MHC Class II DRβ chain is Val, is judged to have resistant tothe onset of the leukemia. As preferred embodiments of the method of thepresent invention, there are provided the aforementioned method which isapplied to a cattle infected by the bovine leukemia virus BLV; theaforementioned method wherein the bovine individual, in which the aminoacid specified by the amino acid number 78 of the β1 domain of thebovine MHC Class II DRβ chain is Val in at least one of the alleles, isjudged to have a resistance to the onset; and the aforementioned methodwherein the bovine individual, in which the amino acid specified by theamino acid number 78 of the β1 domain of the bovine MHC Class II DRβchain is Val in both of the alleles, is judged to have a high resistanceto the onset.

According to another embodiment of the method of the present invention,there is provided a method for judging a resistance to the onset ofbovine leukemia caused by the bovine leukemia virus BLV, which comprisesthe steps of:

(1) amplifying genome DNA isolated from a bovine individual by thepolymerase chain reaction (PCR) to prepare a PCR product containing aDNA coding for a part or full length of the β1 domain of the bovine MHCClass II DRβ chain, and

(2) judging that the bovine individual, in which an amino acidcorresponding to amino acid number 78 of the β1 domain of the bovine MHCClass II DRβ chain is Val in the amino acid sequence encoded by the DNAcontained in the PCR product, has a resistance to the onset of theleukemia. A preferred embodiment of the aforementioned method comprisesa step of digesting the PCR product by using PstI.

According to preferred embodiments of these inventions, there areprovided each of the primer sets set out below, and the aforementionedmethods wherein said primer set is used, preferably in those applied tocattle infected by the bovine leukemia virus BLV. The present inventionfurther provides the following primer sets (1) to (3) each consisting ofA primer and B primer, which are used for judging a possibility of theonset of bovine leukemia caused by the bovine leukemia virus BLV:

Primer set (1)

A primer: 5′-TGTAAAACGACGGCCAGTCTCTCTCTGCAGCACATTTCCT-3′(SEQ ID NO:9)

B primer: 5′-CAGGAAACAGCTATGACCCGCCGCTGCACAGTGAAACTC-3′(SEQ ID NO:9)

Primer set (2)

A primer: 5′-GGAATTCCTCTCTCTGCAGCACATTTCCT-3′(SEQ ID NO:3)

B primer: 5′-AAGTCGACCGCTGCACAGTGAAACTC-3′(SEQ ID NO:4)

Primer set (3)

A primer: a primer which is selected from the group consisting of

5¹-GAGTGTCATTTCTTCAACGGGAC-3′,

5′-GGAGAAGAGTTCGTGCGCTTCGA-3′, and

5′-GGAATTCCTCTCTCTGCAGCACATTTCCT-3′

B primer: 5′-AAGTCGACCGCTGCACAGTGAAACTC-3′

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the structure of the bovine MHC Class II DRβ chain. Inthe figure, (A) shows the structure of bovine MHC Class II DRβ chainmRNA, and (B) shows the full length cDNA coding for the bovine MRC ClassII DRβ chain and the amino acid sequence of the gene product. The β1domain is a portion defined by the amino acid sequence of the amino acidnumber from 1 to 94.

FIGS. 2(A) to (C) show the results of comparison of amino acids of theβ1 domain of the bovine MHC Class II DRβ chain (amino acid sequencesdefined by the amino acid number from 9 to 86) derived from cattleinfected by the bovine leukemia virus BLV but not developing the disease((A): 7 cattle developing lymphocytosis, and (B) and (C): antibodypositive 24 healthy cattle not developing the disease). The numbers atthe left end are ID numbers of bovine individuals, and amino acidsindicated as one letter symbols in the figure.

FIGS. 3 (A) and (B) show the results of comparison of amino acids of theβ1 domain of the bovine MHC Class II DRβ chain (amino acid sequencesdefined by the amino acid number from 9 to 86) derived from cattledeveloping leukemia (24 cattle). The numbers at the left end are IDnumbers of bovine individuals, and amino acids are indicated as oneletter symbols in the figure.

BEST MODE FOR CARRYING OUT THE INVENTION

The method of the present invention is applied to bovine individuals,including cattle infected with the bovine leukemia virus BLV and cattlenot infected with the virus, in order to judge a possibility of theonset of the leukemia of the individuals. Another method of the presentinvention is applied to bovine individuals, including cattle infectedwith the bovine leukemia virus BLV and cattle not infected with thevirus, in order to judge a resistance to the onset of the leukemia ofthe individuals.

According to a preferred embodiment of the present invention, genomicDNA of a bovine individual is isolated, and a gene coding for a part orthe full length of the β1 domain of DRβ chain of the bovine MHC Class II(the second exon of DRB3 gene) is amplified by the PCR method, and thenthe resulting PCR product is subjected to a sequencing to deduce theamino acid sequence defined by the amino acid number from 75 to 78 ofthe β1 domain. When a bovine individual, in which the amino acidsequence (amino acid numbers 75 to 78) is Val-Asp-Thr-Tyr (indicated asVDTY in the one letter symbols), is already with infection by the bovineleukemia virus BLV, or when the individual will suffer from infection bythe bovine leukemia virus BLV, the bovine individual has a possibilityof the onset of the leukemia. Whether or not a bovine individual isinfected by the bovine leukemia virus BLV can be readily verified by atest using an antibody recognizing the bovine leukemia virus BLV.

In order to carry out more accurate judgement, it is preferred tocompare the aforementioned amino acid sequences in the alleles(haplotypes). When the amino acid sequence (amino acid number 75 to 78)is Val-Asp-Thr-Tyr in both of the alleles (i.e., VDTY homozygote), thebovine individual has a high risk of the onset of the leukemia when theindividual is already infected by the bovine leukemia virus BLV, or willsuffer from the infection by the virus. On the other hand, when theamino acid sequences in the alleles are heterozygote of Val-Asp-Thr-Tyr(VDTY) and Val-Asp-Thr-Val (VDTV); heterozygote of Val-Asp-Thr-Tyr(VDTY) and Val-Asp-Arg-Val (VDRV); homozygote of Val-Asp-Thr-Val (VDTV);homozygote of Val-Asp-Arg-Val (VDRV); heterozygote of Val-Asp-Arg-Val(VDRV) and Val-Asp-Thr-Val (VDTV) or the like, the bovine individual hasa very low possibility of the onset of the leukemia even if the bovineindividual is already infected by the bovine leukemia virus BLV or willsuffer from the infection by the virus.

Furthermore, from a viewpoint of a resistance to the onset of heleukemia, the amino acid defined by the amino acid number 78 of the β1domain may be deduced. When a bovine individual having Val (representedas V in the one letter symbol) as the amino acid (i.e., amino acidnumber 78) is already infected by the bovine leukemia virus BLV, or willsuffer from infection by the virus, the bovine individual is resistantto the onset of the leukemia. Also for the judgement of the resistance,it is preferred to compare the aforementioned amino acid in the alleles(haplotypes). When the amino acid, defined by the amino acid number 78of the β1 domain, is Val in at least one of the alleles, the individualhas a resistance to the onset of the leukemia, and when the above aminoacid is Val in both of the alleles, the individual has a high resistanceto the onset of the leukemia.

The amino acid sequence of the β1 domain of the bovine MHC Class II DRβchain was reported by Aida et al. (Aida, Y., et al., Biochem. Biophys.Res. Commun., 209, pp.981-988, 1995). The structure of mRNA of thebovine MHC Class II DRβ chain (A), and the full length cDNA and theamino acid sequence of the gene product (B) are shown in FIG. 1. In thefigure, the β1 domain is a portion defined by the amino acid sequence ofamino acid number from 1 to 94, and the nucleotide sequence and theamino acid sequence are shown where the peptide sequence of the aminoacid number from 75 to 78 is “Val-Asp-Thr-Tyr (VDTY)”.

Cattle to be judged by the method according to the present invention arenot particularly limited. The method may be applied to any sorts ofcattle including dairy cattle, dairy and beef cattle, beef cattle,working cattle, working and beef cattle and the like, so long as theymay be infected by the bovine leukemia virus BLV and have a possibilityof developing the leukemia owing to the infection. More specifically,examples include Japanese cattle such as Japanese Black and JapaneseShorthorn, or breeds such as Holstein, Jersey, Hereford, Aberdeen Angus,and Friesian. However, breeds are not limited to these examples.

As a sample for preparing genomic DNA from bovine individuals,peripheral blood, organ and the like can be utilized. For example, atissue section of the lymph node and other may be used as the organ. Asmethods for preparing genomic DNA from the sample mentioned above, anymethods available to those skilled in the art can be employed. Whenperipheral blood leucocytes or peripheral blood lymphocytes are used asa sample, for example, the method of Hughes et al. (Hughes, S. H., Cell,15, pp.1397-1410, 1978) may be applied. When an organ is used, forexample, a frozen tissue section may be sliced by using scissors, andthen treated by the sodium dodecylsulfate and phenol-chloroform method(Mcknight, G. S., Cell, 14, pp.403-413 1978) to obtain genomic DNA. Thesimplified extraction of genomic DNA from cells may also be used, whosedetails are described in the examples.

As primers used for amplifying the resulting genomic DNA by the PCRmethod, any primers may be used so long as they can amplify a DNAcontaining a gene coding for a partial amino acid sequence of amino acidnumber from 75 to 78 of the β1 domain of the DRβ chain of the bovine MHCClass II or the full length of the β1 domain.

An example of a primer set most suitably used for the methods of thepresent invention includes primer set (1):

A primer: 5′-TGTAAAACGACGGCCAGTCTCTCTCTGCAGCACATTTCCT-3′; and

B primer: 5′-CAGGAAACAGCTATGACCCGCCGCTGCACAGTGAAACTC-3′

which enables direct sequencing methods such as the cycle sequencing andthe Dynabeads DNA direct sequencing. As primer sets introduced with arestriction endonuclease cleavage site, primer set (2):

A primer: 5′-GGAATTCCTCTCTCTGCAGCACATTTCCT-3′; and

B primer: 5′-AAGTCGACCGCTGCACAGTGAAACTC-3′,

or primer set (3):

A primer: a primer selected from the group consisting of:

5′-GAGTGTCATTTCTTCAACGGGAC-3′,

5′-GGAGAAGAGTTCGTGCGCTTCGA-3′, and

5′-GGAATTCCTCTCTCTGCAGCACATTTCCT-3′; and

B primer: 5′-AAGTCGACCGCTGCACAGTGAAACTC-3′

may be utilized. In particular, by digesting PCR alleles with PstI thatare amplified by using the primer set (3), and then observingdifferences in the resulting cleavage patterns, it can easily judgewhether or not the bovine individual is resistant to the leukemia, orwhether or not the individual has a possibility of the onset of theleukemia. However, primers and primer sets which may be used for themethods of the present invention are not limited to the forgoingexamples.

An amount of DNA used for the PCR method can be appropriately chosen.For example, the amount may be about 0.1-0.5 μg when peripheral bloodleucocytes or peripheral lymphocytes are used. As sequencing methodsapplied to the DNA amplified as descried above (the PCR product), anymethods available to those skilled in the art may be utilized. Forexample, the direct sequencing may preferably be used, whose specificexamples are described in the examples. Most of cattle areheterozygotes, and when alleles derived from father and mother cattlemay have different nucleotide sequences, the direct sequencing may failto determine which of the alleles corresponds to the target sequence. Inthat case, the PCR product amplified by using the above primer set (2)may be digested with restriction endonuclease EcoRI and Sal I, and thensubcloned into a vector to carry out the sequencing of only one of thealleles, and the results may be referred to for comparison to enable adefinite sequencing of the other allele. To obtain more precise geneticinformation, it is preferred that both of the alleles from the PCRproduct are subcloned and each of the nucleotide sequences isdetermined. The specific method and applicable primers are detailed inthe following examples.

EXAMPLES

The present invention will be explained more specifically by referringto examples. However, the scope of the present invention is not limitedto the examples set out below.

Example 1

Examination of a Possibility of the Onset of the Leukemia

Peripheral blood was collected as a sample from a bovine individual byusing a syringe containing an anticoagulant, and centrifuged underconditions of 4° C. and 3,000 rpm for 20 minutes to obtain a leucocytelayer. The separated leucocyte layer was washed with phosphate bufferedsaline (PBS) and centrifuged to obtain a pellet, which was used as asample of peripheral blood leucocyte. Peripheral blood lymphocytes werealso obtained by the method of Miyasaka et al. (Miyasaka, M. and Trnka,Z., Immunological Methods, Vol.3, pp.403-423, 1985, Academic Press, NY)from peripheral blood obtained in the same manner as described above,and a sample of peripheral lymphocyte was prepared by obtaining a pelletas described above. A BLV infected cell suspension was centrifuged underconditions of 4° C. and 1,100 rpm for 5 minutes to remove a culturemedium, and the cells were washed with PBS and centrifuged to obtain apellet as a sample. In addition, tissue sections were isolated from thelymph node and a tumor tissue of a cattle which developed BLV infectionlymphosarcoma, and rapidly frozen in liquid nitrogen withoutimmobilization, and then stored at −80° C. as samples of the tissuesections.

Each of the above sample cells were washed twice with PBS in a 1.5ml-microcentrifugal tube, and the precipitated cells were suspendedagain in PBS by using a vortex mixer. To 1×10⁶ cells, 200 μl of 1×PCRbuffer [10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, 0.5% Tween-20]and 1 μl of Proteinase K (20 mg/ml) were added, and the cells weresuspended again by a vortex mixer and incubated at 56° C. for 45-60minutes. The mixture was further treated at 95° C. for ten minutes, andcooled on ice for 5 minutes or more. About 5-10 μl of the reactionmixture was used for amplification by PCR.

The genome DNA was dissolved in 50 μl of 1×PCR buffer [10 mM Tris-HCl(pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.001% (w/v) gelatin] containing 200μM of each dNTP, 0.2-0.4 μM of primers, and 2.5 units of Taq polymerase(Gene Amp Kit; Perkin-Elmer Cetus), and then subjected to amplificationby 25 cycles, each cycle consisting of treatments at 94° C. for 1minute, at 61° C for 1 minute, and at 72° C. for 1 minute, and thenfurther treated at 72° C. for 5 minutes. As the primers, the followingprimers were used:

A primer: 5′-TGTAAAACGACGGCCAGTCTCTCTCTGCAGCACATTTCCT-3′

B primer: 5′-CAGGAAACAGCTATGACCCGCCGCTGCACAGTGAAACTC-3′

which can specifically amplify the β1 domain of the bovine MHC Class IIDRβ chain (β1 domain of BoLA-DRβ: the second exon of DRB3 gene) by thePCR method. Specific biotinylation was introduced into the 5′ end of theB primer. These primers can be suitably used for the cycle sequencing.

20 μl of DYNABEADS M-280 Streptoavidin (Dynal A. S, N-0212, Oslo,Norway) was washed with 100 μl of 2×binding-washing buffer (B&W buffer:10 mM Tris-HCl (pH 7.5), 1.0 mM EDTA, 2M NaCl, 0.1% Tween-20), and thebeads were suspended again in 80 μl of 2×B&W buffer. The above PCRproduct (50 μl) was added to the bead suspension and gently mixed bypipetting, and then incubated at room temperature for 15 minutes withslow rotation using a wheel rotator. The tube containing the immobilizedPCR product was put on a magnet (Dynal MPC) and the supernatant wasremoved by using a pipet, and then 100 μl of 2×B&W buffer was added towash the beads. The supernatant was removed again by using a magnet, andthe residue was suspended in 50 μl of 0.1 M NaOH prepared just beforeuse.

The beads immobilizing the biotinylated chains were gathered on the tubewall by using a magnet and the supernatant was removed, and then thebeads were washed once with 50 μl of 0.1 M NaOH and three times with 100μl of 1×B&W buffer, and once with 50 μl of TE buffer. In everyoperation, the beads were resuspended with smooth strokes. After washingwith 100 μl of distilled water, the supernatant was removed, anddistilled water was added to the residue to adjust the volume for theuse in a sequencing. The sequencing was performed by using BcaBESTDideoxy Sequencing Kit (Takara Biomedicals) and according to theconditions described in the attached instructions. The following primerswere used as sequencing primers.

Forward primer: 5′-TGTAAAACGACGGCCAGT-3′

Reverse primer: 5′-CAGGAAACAGCTATGACC-3′

The results are shown in FIGS. 2 and 3 (in the figures, amino acids ofnumber 9 to 86 of the β1 domains of the bovine MHC Class II DRβ areshown, and the numbers at the left end are ID numbers of bovineindividuals). By comparing the amino acids of the β1 domain of bovineMHC Class II DRβ derived from cattle infected by the bovine leukemiavirus but not developing the leukemia [7 cattle with lymphocytosis(pre-cancer state), and 24 cattle not developing the leukemia (antibodypositive healthy cattle not developing the disease), top and bottom ofFIG. 2, respectively], and cattle already developing the leukemia (24cattle, FIG. 3), a markedly characteristic result was obtained that thecattle with the developed leukemia had Val-Asp-Thr-Tyr (VDTY) motif asthe sequence of amino acid number from 75 to 78 in both of the alleles.The portion of the amino acids from 75 to 78 is located on an α-helix ofthe β1 domain, and may have a function as a T cell recognition site.Furthermore, as a result of an analysis using a computer, it wasrevealed that this motif exists only in pol protein in the bovineleukemia virus BLV.

The above results are summarized in Table 1. The representation of thegenotype such as VDTY/VDTY in the table indicates amino acid sequencesof the both allele (amino acid number 75 to 78 of the β1 domain of thebovine MHC Class II DRβ chain) described as the one letter symbols. Theinfection status of the BLV infected cattle were classified according tothe criteria of Levy et al. (Levy, D., et al., Int. J. Cancer, 19,pp.822-827, 1977) and Aida et al. (Aida, Y., et al., Cancer Res., 52,pp.6463-6470, 1992).

TABLE 1 BLV infection status (positive rate) Development of Genotypeleukemia Lymphocytosis Healthy VDTY/VDTY 19/24  5/7 4/24 VDTY/VDTV 2/242/7 2/24 VDTY/VDRV 2/24 0/7 14/24  VDRV/VDRV 0/24 0/7 1/24 VDTV/VDTV1/24 0/7 0/24 VDTV/VDRV 0/24 0/7 3/24

Example 2

Study on Resistance to the Onset of Leukemia

It the same manner as in Example 1, kinds of the amino acid at number 78of the β1 domains of bovine MHC Class II DRβ was determined for cattledeveloping the leukemia (24 cattle), cattle not developing the leukemia(cattle with lymphocytosis and healthy cattle, 31 individuals in total),and the results are shown in Table 2. The kind of the amino acids atnumbers 71 and 74 were also determined (in the table, amino acids areindicated as one letter symbols, Y: Tyr; V: Val; R: Arg; E: Glu; K: Lys;and N: Asn). As a result, it was revealed that individuals where the78th amino acid was heterozygote of valine and tyrosine, and individualswhere the 78th amino acid was homozygote of valine were resistant to theonset of the leukemia, and in particular, the individuals where the 78thamino acid was homozygote of valine were highly resistant to the onsetof the leukemia. Furthermore, because all of the 74th amino acids ofcattle not developing the leukemia were Gln or Asn, and the 71st aminoacid residues were Lys or Arg, it was suggested that individuals havingthe allele where the 71st amino acid is lysine or arginine, the 74thamino acid is glutamic acid or asparagine, and the 78th amino acid isvaline have high resistant to the onset of the leukemia.

TABLE 2 Positive Genotype BLV infection status rate Y⁷⁸/Y⁷⁸ Cattledeveloping leukemia 19/24 V⁷⁸/Y⁷⁸ Cattle developing leukemia  4/24(R⁷¹-E⁷⁴-V⁷⁸/Y⁷⁸:  3/24) (K⁷¹-E⁷⁴-V⁷⁸/Y⁷⁸:  1/24) (K⁷¹-N⁷⁴-V⁷⁸/Y⁷⁸: 0/24) V⁷⁸/V⁷⁸ Cattle developing leukemia  1/24(R⁷¹-E⁷⁴-V⁷⁸/R⁷¹-E⁷⁴-V⁷⁸:  1/24) (K⁷¹-E⁷⁴-V⁷⁸/K⁷¹-E⁷⁴-V⁷⁸:  0/24)(K⁷¹-N⁷⁴-V⁷⁸/K⁷¹-N⁷⁴-V⁷⁸:  0/24) Y⁷⁸/Y⁷⁸ Cattle not developing leukemia 9/31 V⁷⁸/Y⁷⁸ Cattle not developing leukemia 18/31 (R⁷¹-E⁷⁴-V⁷⁸/Y⁷⁸:11/31) (K⁷¹-E⁷⁴-V⁷⁸/Y⁷⁸:  4/31) (K⁷¹-N⁷⁴-V⁷⁸/Y⁷⁸:  3/31) V⁷⁸/V⁷⁸ Cattlenot developing leukemia  4/31 (R⁷¹-E⁷⁴-V⁷⁸/R⁷¹-E⁷⁴-V⁷⁸:  3/31)(K⁷¹-E⁷⁴-V⁷⁸/K⁷¹-E⁷⁴-V⁷⁸:  1/31) (K⁷¹-N⁷⁴-V⁷⁸/K⁷¹-N⁷⁴-V⁷⁸:  0/31)

Example 3

Method for Quick Judgement of Possibility and Resistance to the Onset

As described above, individuals having the gene coding for Val as theamino acid at number 78 of the β1 domain of the bovine MHC Class II DRβchain are resistant to the leukemia caused by the bovine leukemia virus,whereas individuals where the 78th amino acid is Tyr in both of thealleles have a possibility of the onset of the leukemia. Therefore,whether or not an bovine individual is leukemia resistant, or whether ornot an individual has a possibility of the onset of the leukemia iseasily judged by utilizing restriction endonuclease PstI cleavage sitewhich is present in a gene where the 78th allele is Val but absent in agene where the 78th allele is Tyr, i.e., by digesting PCR amplifiedalleles and differentiating the cleavage pattern.

The following primers were used as PCR primers.

A primer

DRB40: 5′-GAGTGTCATTTCTTCAACGGGAC-3′

DRB100: 5′-GGAGAAGAGTTCGTGCGCTTCGA-3′

ERB3: 5′-GGAATTCCTCTCTCTGCAGCACATTTCCT-3′

B primer

SRB3: 5′-AAGTCGACCGCTGCACAGTGAAACTC-3′

The conditions of the PCR were similar to the conditions of Example 1.Specifically, amplification was performed by 35 cycles, each cycleconsisting of the following steps depending on the combination of theprimers, followed by a treatment at 72° C. for 10 minutes. The genomicDNA was used in an amount of 100 ng for 100 μl of the PCR system.

DRB40/SRB3: 94° C. for 1 minute, 63° C. for 2 minutes, 72° C. for 2minutes

DRB100/SRB3: 94° C. for 1 minute, 66° C. for 2 minutes, 72° C. for 2minutes

ERB3/SRB3: 94° C. for 1 minute, 61° C. for 2 minutes, 72° C. for 2minutes

The PCR product was subjected to 2% agarose gel electrophoresis, andthen cleaved by using restriction endonuclease PstI (1.2 μl of10×restriction endonuclease buffer, 6-7 μl of DNA after amplification, 2units of restriction endonuclease PstI, and H₂O in the total volume of12 μl). After completion of the reaction with the restrictionendonuclease, each specimen was examined by 3% agarose gelelectrophoresis for judgement. The results are shown in Table 3.

TABLE 3 PCR Size (bp) of PstI fragment product Allele for Allele forPrimer (bp) Y V Y/Y Y/V DRB40/SRB3 247 199, 48 247 199, 48 247, 199, 48DRB100/SRB3 187 139, 48 187 139, 48 139, 187, 48 ERB3/SRB3* 292 226, 48274 226, 48 226, 274, 48 *PstI cleavage site is present in the ERB3primer, and hence a 18 bp fragment was contained in each reactionmixture.

Industrial Applicability

A possibility of the onset of the leukemia caused by the bovine leukemiavirus (BLV) and a resistance thereto of a bovine individual can besurely estimated by the methods of the present invention. Therefore, theinvention enables safe cattle breeding and achieves prevention ofeconomic loss of stockbreeders.

115 1 40 DNA Artificial Sequence Description of Artificial SequencePRIMER FOR JUDGING POSSIBILITY OF ONSET OF BOVINE LEUKEMIA 1 tgtaaaacgacggccagtct ctctctgcag cacatttcct 40 2 39 DNA Artificial SequenceDescription of Artificial Sequence PRIMER FOR JUDGING POSSIBILITY OFONSET OF BOVINE LEUKEMIA 2 caggaaacag ctatgacccg ccgctgcaca gtgaaactc 393 29 DNA Artificial Sequence Description of Artificial Sequence PRIMERFOR JUDGING POSSIBILITY OF ONSET OF BOVINE LEUKEMIA 3 ggaattcctctctctgcagc acatttcct 29 4 26 DNA Artificial Sequence Description ofArtificial Sequence PRIMER FOR JUDGING POSSIBILITY OF ONSET OF BOVINELEUKEMIA 4 aagtcgaccg ctgcacagtg aaactc 26 5 23 DNA Artificial SequenceDescription of Artificial Sequence PRIMER FOR JUDGING POSSIBILITY OFONSET OF BOVINE LEUKEMIA 5 gagtgtcatt tcttcaacgg gac 23 6 23 DNAArtificial Sequence Description of Artificial Sequence PRIMER FORJUDGING POSSIBILITY OF ONSET OF BOVINE LEUKEMIA 6 ggagaagagt tcgtgcgcttcga 23 7 29 DNA Artificial Sequence Description of Artificial SequencePRIMER FOR JUDGING POSSIBILITY OF ONSET OF BOVINE LEUKEMIA 7 ggaattcctctctctgcagc acatttcct 29 8 26 DNA Artificial Sequence Description ofArtificial Sequence PRIMER FOR JUDGING POSSIBILITY OF ONSET OF BOVINELEUKEMIA 8 aagtcgaccg ctgcacagtg aaactc 26 9 4 PRT BOVINE 9 Val Asp ThrTyr 1 10 1141 DNA BOVINE 10 ctctgctgtt ctccggcatg gtgtgcctgt atttctctggaggctcctgg atggcagctc 60 tgatagtgat gctgatggtg ctgtgccctc ccctggcctgggccagggag atccaaccac 120 atttcctgga gtataccaag aaagagtgtc atttcttcaacgggaccgag cgggtgcggt 180 tcctggacag atacttccat aatggagaag agttcgtgcgcttcgatagc gactggggcg 240 agtaccgggc ggtgaccgag ctagggcggc cggacgccaagtactggaac agccagaagg 300 acttcctgga ggagaagcgg gccgcggtgg acacgtactgcagacacaac tacggggtcg 360 gtgagagttt cactgtgcag cggcgagtgg aacctatagtgactgtgtat cctgcaaaga 420 cccagcccct gcagcaccac aacctcctgg tctgctctgtgaacggtttc tacccaggca 480 acattgaagt caggtggttc cggaatggcc atgaagaggaggctggggtg atctccacag 540 gcctgatcca gaatggagac tggaccttcc agaccatggtgatgcttgaa acagttcctc 600 agagtggaga ggtctacacc tgccaagtgg agcaccccagccagacaagc cctatcacag 660 tagaatggag ggcacggtct gactctgctc agagcaagatgatgagtgga gtcgggggct 720 tcgttctggg tctgttcttc cttgccgtgg ggctcttcatctacttcagg aatcagaaag 780 gacgccctac acttcagcca acagggctcc tgagctgaagtgaagatggt cacactcaag 840 gaagaacctt ctgtcccagc ttcttcacag catggaaaggtttcctgctt agtgctaact 900 cttccacaat gaagtacttt ctcaggatct catttgctcctggctcagtg accccttaaa 960 aactgtcctc gatggttttc tcagtcacct ccaccctgctgccctcagcc tttgacctgg 1020 aagttctcaa tattgattcc agtaccttat gttctttcttccttggttcc ctttcttttc 1080 aacttctgtt tcctgtgcat ctgagctcat ctgttcattttactttataa tgtgttctct 1140 c 1141 11 266 PRT BOVINE 11 Met Val Cys LeuTyr Phe Ser Gly Gly Ser Trp Met Ala Ala Leu Ile 1 5 10 15 Val Met LeuMet Val Leu Cys Pro Pro Leu Ala Trp Ala Arg Glu Ile 20 25 30 Gln Pro HisPhe Leu Glu Tyr Thr Lys Lys Glu Cys His Phe Phe Asn 35 40 45 Gly Thr GluArg Val Arg Phe Leu Asp Arg Tyr Phe His Asn Gly Glu 50 55 60 Glu Phe ValArg Phe Asp Ser Asp Trp Gly Glu Tyr Arg Ala Val Thr 65 70 75 80 Glu LeuGly Arg Pro Asp Ala Lys Tyr Trp Asn Ser Gln Lys Asp Phe 85 90 95 Leu GluGlu Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr 100 105 110 GlyVal Gly Glu Ser Phe Thr Val Gln Arg Arg Val Glu Pro Ile Val 115 120 125Thr Val Tyr Pro Ala Lys Thr Gln Pro Leu Gln His His Asn Leu Leu 130 135140 Val Cys Glu Val Asn Gly Phe Tyr Pro Gly Asn Ile Glu Val Arg Trp 145150 155 160 Phe Arg Asn Gly His Glu Glu Glu Ala Gly Val Ile Ser Thr GlyLeu 165 170 175 Ile Gln Asn Gly Asp Trp Thr Phe Gln Thr Met Val Met LeuGlu Thr 180 185 190 Val Pro Gln Ser Gly Glu Val Tyr Thr Cys Gln Val GluHis Pro Ser 195 200 205 Gln Thr Ser Pro Ile Thr Val Glu Trp Arg Ala ArgSer Asp Ser Ala 210 215 220 Gln Ser Lys Met Met Ser Gly Val Gly Gly PheVal Leu Gly Leu Phe 225 230 235 240 Phe Leu Ala Val Gly Leu Phe Ile TyrPhe Arg Asn Gln Lys Gly Arg 245 250 255 Pro Thr Leu Gln Pro Thr Gly LeuLeu Ser 260 265 12 94 PRT BOVINE 12 Arg Glu Ile Gln Pro His Phe Leu GluTyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Phe Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg PheAsp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg ProAsp Ala Lys Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Glu Lys ArgAla Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly GluSer Phe Thr Val Gln Arg Arg 85 90 13 94 PRT BOVINE 13 Arg Glu Ile GlnPro His Phe Leu Glu Tyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Phe Leu Asn Arg Tyr Phe His 20 25 30 Asn Gly GluGlu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu IleLeu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His AsnTyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 14 94 PRT BOVINE14 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Thr Lys Ser Glu Cys His 1 510 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Tyr Thr 2025 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 3540 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Gln Trp Asn Ser Gln 5055 60 Lys Asp Phe Leu Glu Ser Arg Arg Thr Ala Val Asp Thr Tyr Cys Arg 6570 75 80 His Asn Tyr Gly Val Phe Glu Ser Phe Thr Val Gln Arg Arg 85 9015 94 PRT BOVINE 15 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Cys Lys ArgGlu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu AspArg Tyr Ser Tyr 20 25 30 Asn Gly Lys Glu Arg Val Arg Phe Asp Ser Asp TrpGly Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Ser Ala Glu TyrTrp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Gln Arg Arg Ala Ala Val AspThr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Val Glu Ser Phe Thr ValGln Arg Arg 85 90 16 94 PRT BOVINE 16 Arg Glu Ile Gln Pro His Phe LeuGln Tyr His Lys Gly Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu ArgVal Arg Leu Leu Asp Arg His Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val ArgPhe Asp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgPro Ser Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg ArgArg Ala Glu Val Asp Thr Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val ValGlu Ser Phe Thr Val Gln Arg Arg 85 90 17 94 PRT BOVINE 17 Arg Glu IleGln Pro His Phe Leu Glu Tyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe PheAsn Gly Thr Asp Arg Val Arg Phe Leu Asn Arg Tyr Phe His 20 25 30 Asn GlyGlu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala ValThr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys GluIle Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 HisAsn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 18 94 PRTBOVINE 18 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Tyr Leu Asp Arg TyrPhe His 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly GluTyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Arg Val Ala Glu Gln Leu AsnGly Gln 50 55 60 Lys Asp Thr Leu Glu Arg Glu Arg Ala Tyr Val Asp Thr TyrCys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln ArgArg 85 90 19 94 PRT BOVINE 19 Arg Glu Ile Gln Pro His Phe Leu Glu TyrSer Thr Ser Glu Cys His 1 5 10 15 Phe Ser Asn Gly Thr Glu Arg Val ArgLeu Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe AspSer Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Ala Glu Leu Gly Arg Pro AspAla Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Ser Arg Arg ThrAla Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Ile Glu SerPhe Thr Val Gln Arg Arg 85 90 20 94 PRT BOVINE 20 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Cys Lys Arg Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Asp Arg Cys Phe His 20 25 30 Asn Gly Glu GluPhe Val Arg Phe Asp Ser Asp Arg Gly Glu Phe Arg 35 40 45 Ala Val Thr GluLeu Gly Arg Arg Val Ala Glu Gln Trp Asn Ser Gln 50 55 60 Lys Asp Phe LeuGlu Glu Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn TyrGly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 21 94 PRT BOVINE 21Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Thr Lys Lys Glu Cys His 1 5 1015 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asn Arg Tyr Phe His 20 2530 Asn Gly Glu Glu Phe Val Arg Phe Gly Ser Asp Trp Gly Glu Tyr Arg 35 4045 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 5560 Lys Glu Ile Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 7075 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 2294 PRT BOVINE 22 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser GluCys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp ArgTyr Phe His 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp Trp GlyGlu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Gln Arg Val Ala Glu Tyr CysAsn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala Arg Ala Ala Val Asp ThrTyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val GlnArg Arg 85 90 23 94 PRT BOVINE 23 Arg Glu Ile Gln Pro His Phe Leu GluTyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly Glu Glu Thr Val Arg PheAsp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg GlnAsp Ala Glu Gln Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala ArgAla Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 Tyr Asn Tyr Gly Val Gly GluSer Phe Thr Val Gln Arg Arg 85 90 24 94 PRT BOVINE 24 Arg Glu Ile GlnPro His Phe Leu Gln Tyr His Lys Gly Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Leu Leu Asp Arg His Phe Tyr 20 25 30 Asn Gly GluGlu Tyr Val Arg Phe Asp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Pro Ser Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp PheLeu Glu Arg Arg Arg Ala Glu Val Asp Thr Val Cys Arg 65 70 75 80 His AsnTyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 25 94 PRT BOVINE25 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 510 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Glu Arg Ser Phe Tyr 2025 30 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 3540 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 5055 60 Lys Glu Ile Leu Glu Glu Arg Arg Ala Glu Val Asp Arg Val Cys Arg 6570 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 9026 94 PRT BOVINE 26 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys SerGlu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu GluArg Ser Phe Tyr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp TrpGly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu TyrTrp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg Lys Arg Ala Asn Val AspArg Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr ValGln Arg Arg 85 90 27 94 PRT BOVINE 27 Arg Glu Ile Gln Pro His Phe LeuGlu Tyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu ArgVal Arg Phe Leu Asn Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val ArgPhe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgPro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg AlaArg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val GlyGlu Ser Phe Thr Val Gln Arg Arg 85 90 28 94 PRT BOVINE 28 Arg Glu IleGln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe PheAsn Gly Thr Glu Arg Val Arg Phe Leu Glu Arg Ser Phe Tyr 20 25 30 Asn GlyGlu Glu Asn Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala ValThr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys GluTyr Leu Glu Glu Arg Arg Ala Glu Val Asp Arg Val Cys Arg 65 70 75 80 HisAsn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 29 94 PRTBOVINE 29 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Glu Arg SerPhe Tyr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp Gly GluTyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp AsnSer Gln 50 55 60 Lys Glu Ile Leu Glu Arg Lys Arg Ala Asn Val Asp Arg ValCys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln ArgArg 85 90 30 94 PRT BOVINE 30 Arg Glu Ile Gln Pro His Phe Leu Glu TyrThr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val ArgPhe Leu Asn Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe AspSer Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro AspAla Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg Ala Arg AlaAla Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Ile Glu SerPhe Thr Val Gln Arg Arg 85 90 31 94 PRT BOVINE 31 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Cys Lys Arg Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Asp Arg Cys Phe His 20 25 30 Asn Gly Glu GluPhe Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Thr GluLeu Gly Arg Arg Val Ala Glu Gln Trp Asn Ser Gln 50 55 60 Lys Asp Phe LeuGlu Glu Arg Arg Ala Glu Val Asp Arg Val Cys Arg 65 70 75 80 His Asn TyrGly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 32 94 PRT BOVINE 32Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Thr Lys Lys Glu Cys His 1 5 1015 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asn Arg Tyr Phe His 20 2530 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 4045 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 5560 Lys Glu Ile Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 7075 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 3394 PRT BOVINE 33 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Cys Lys Ser GluCys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Glu ArgSer Phe Tyr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp GlyGlu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr TrpAsn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Glu Arg Arg Ala Glu Val Asp ArgVal Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val GlnArg Arg 85 90 34 94 PRT BOVINE 34 Arg Glu Ile Gln Pro His Phe Leu GlnTyr His Lys Gly Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Leu Leu Asp Arg His Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val Arg PheAsp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg ProSer Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Arg ArgAla Glu Val Asp Thr Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val Val GluSer Phe Thr Val Gln Arg Arg 85 90 35 94 PRT BOVINE 35 Arg Glu Ile GlnPro His Phe Leu Glu Tyr Leu Lys Ser Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Phe Leu Glu Arg Ser Phe Tyr 20 25 30 Asn Gly GluGlu Asn Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Pro Asp Ala Lys Tyr Trp Asn Ser Gln 50 55 60 Lys Asp LeuLeu Glu Arg Lys Arg Ala Asn Val Asp Thr Tyr Cys Arg 65 70 75 80 His AsnTyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 36 94 PRT BOVINE36 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Cys Lys Arg Glu Cys His 1 510 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Cys Phe His 2025 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 3540 45 Ala Val Thr Glu Leu Gly Arg Arg Val Ala Glu Gln Trp Asn Ser Gln 5055 60 Lys Asp Phe Leu Glu Glu Arg Arg Ala Glu Val Asp Arg Val Cys Arg 6570 75 80 His Asn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 9037 94 PRT BOVINE 37 Arg Glu Ile Gln Pro His Phe Leu Gln Tyr His Lys GlyGlu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu AspArg His Phe Tyr 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp TrpAsp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Ala Ala Glu GlnTrp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Gln Lys Arg Ala Glu Val AspArg Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr ValGln Arg Arg 85 90 38 94 PRT BOVINE 38 Arg Glu Ile Gln Pro His Phe LeuGln Tyr His Lys Gly Glu Cys His 1 5 10 15 Phe Leu Asn Gly Thr Glu ArgVal Arg Leu Leu Asp Arg His Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val ArgPhe Asp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgPro Ser Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg ArgArg Ala Glu Val Asp Thr Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val GlyGlu Ser Phe Thr Val Gln Arg Arg 85 90 39 94 PRT BOVINE 39 Arg Glu IleGln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe PheAsn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn GlyGlu Glu Thr Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala ValThr Glu Leu Gly Arg Gln Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys AspPhe Leu Glu Glu Lys Arg Ala Glu Val Asp Arg Val Cys Arg 65 70 75 80 HisAsn Tyr Gly Gly Met Glu Ser Phe Thr Val Gln Arg Arg 85 90 40 94 PRTBOVINE 40 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Glu Arg SerPhe Tyr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp Gly GluTyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp AsnSer Gln 50 55 60 Lys Glu Ile Leu Glu Arg Lys Arg Ala Asn Val Asp Arg ValCys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln ArgArg 85 90 41 94 PRT BOVINE 41 Arg Glu Ile Gln Pro His Phe Leu Glu TyrThr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val ArgPhe Leu Asn Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe AspSer Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro AspAla Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg Ala Arg AlaAla Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu SerPhe Thr Val Gln Arg Arg 85 90 42 94 PRT BOVINE 42 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Asp Arg Ser Phe Tyr 20 25 30 Asn Gly Glu GluAsn Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr GluLeu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile LeuGlu Glu Arg Arg Ala Glu Val Asp Arg Val Cys Arg 65 70 75 80 His Asn TyrGly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 43 94 PRT BOVINE 43Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Thr Lys Lys Glu Cys His 1 5 1015 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe His 20 2530 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 4045 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 5560 Lys Glu Ile Leu Glu Glu Arg Arg Ala Glu Val Asp Arg Val Cys Arg 65 7075 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 4494 PRT BOVINE 44 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Thr Lys Lys GluCys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asn ArgTyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp GlyGlu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr TrpAsn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg Ala Arg Ala Ala Val Asp ThrTyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val GlnArg Arg 85 90 45 94 PRT BOVINE 45 Arg Glu Ile Gln Pro His Phe Leu GluTyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly Glu Glu Asn Val Arg PheAsp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg ProAsp Ala Glu Gln Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Ser Arg ArgThr Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Phe GluSer Phe Thr Val Gln Arg Arg 85 90 46 94 PRT BOVINE 46 Arg Glu Ile GlnPro His Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Phe Leu Asp Arg Ser Phe Tyr 20 25 30 Asn Gly GluGlu Asn Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp IleLeu Glu Glu Arg Arg Ala Glu Val Asp Arg Val Cys Arg 65 70 75 80 His AsnTyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 47 94 PRT BOVINE47 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 510 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Tyr Leu Asp Arg Tyr Phe His 2025 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 3540 45 Ala Val Thr Glu Leu Gly Arg Arg Val Ala Glu Gln Leu Asn Gly Gln 5055 60 Lys Asp Thr Leu Glu Arg Glu Arg Ala Tyr Val Asp Thr Tyr Cys Arg 6570 75 80 His Asn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 9048 94 PRT BOVINE 48 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys SerGlu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu AspArg Tyr Tyr Thr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp TrpGly Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu GlnTrp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Ser Arg Arg Ala Ala Val AspThr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Phe Glu Ser Phe Thr ValGln Arg Arg 85 90 49 94 PRT BOVINE 49 Arg Glu Ile Gln Pro His Phe LeuGlu Tyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu ArgVal Arg Phe Leu Asn Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val ArgPhe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgPro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg AlaArg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val GlyGlu Ser Phe Thr Val Gln Arg Arg 85 90 50 94 PRT BOVINE 50 Arg Glu IleGln Pro His Phe Leu Glu Tyr Ala Thr Ser Glu Cys His 1 5 10 15 Phe PheAsn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe His 20 25 30 Asn GlyGlu Glu Leu Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala ValThr Glu Leu Gly Arg Pro Ser Ala Val Pro Trp Asn Ser Gln 50 55 60 Lys AspPhe Leu Glu Gly Glu Arg Ala Ser Val Asp Thr Tyr Cys Arg 65 70 75 80 HisAsn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 51 94 PRTBOVINE 51 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Cys Lys Arg Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg TyrPhe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp Trp Gly GluPhe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp AsnSer Gln 50 55 60 Lys Asp Phe Leu Glu Glu Arg Arg Ala Glu Val Asp Arg ValCys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln ArgArg 85 90 52 94 PRT BOVINE 52 Arg Glu Ile Gln Pro His Phe Leu Gln TyrHis Lys Gly Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val ArgLeu Leu Asp Arg His Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe AspSer Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro SerAla Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Arg Arg AlaGlu Val Asp Thr Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val Val Glu SerPhe Thr Val Gln Arg Arg 85 90 53 94 PRT BOVINE 53 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Glu Arg Ser Phe Tyr 20 25 30 Asn Gly Glu GluAsn Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr GluLeu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile LeuGlu Glu Arg Arg Ala Glu Val Asp Arg Val Cys Arg 65 70 75 80 His Asn TyrGly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 54 94 PRT BOVINE 54Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 5 1015 Phe Phe Asn Gly Thr Glu Arg Val Arg Tyr Leu Asp Arg Tyr Phe His 20 2530 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 4045 Ala Val Thr Glu Leu Gly Arg Arg Val Ala Glu Gln Leu Asn Gly Gln 50 5560 Lys Asp Thr Leu Glu Arg Glu Arg Ala Tyr Val Asp Thr Tyr Cys Arg 65 7075 80 His Asn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 5594 PRT BOVINE 55 Arg Glu Ile Gln Pro His Phe Leu Gln Tyr His Lys Gly GluCys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Asp ArgHis Phe Tyr 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp AspGlu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Ala Ala Glu Gln TrpAsn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Gln Lys Arg Ala Glu Val Asp ArgVal Cys Arg 65 70 75 80 His Asn Tyr Gly Gly Val Glu Ser Phe Thr Val GlnArg Arg 85 90 56 94 PRT BOVINE 56 Arg Glu Ile Gln Pro His Phe Leu GluTyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Phe Leu Asn Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg PheAsp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg ProAsp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Ile Leu Glu Arg Ala ArgAla Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly GluSer Phe Thr Val Gln Arg Arg 85 90 57 94 PRT BOVINE 57 Arg Glu Ile GlnPro His Phe Leu Gln Tyr His Lys Gly Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Leu Leu Asp Arg His Phe Tyr 20 25 30 Asn Gly GluGlu Tyr Val Arg Phe Asp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Pro Ser Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp PheLeu Glu Arg Arg Arg Ala Glu Val Asp Thr Val Cys Arg 65 70 75 80 His AsnTyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 58 94 PRT BOVINE58 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 510 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Leu Leu Asp Arg Tyr Phe His 2025 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 3540 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 5055 60 Lys Glu Ile Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 6570 75 80 His Asn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 9059 94 PRT BOVINE 59 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Thr Lys LysGlu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu AspArg Tyr Tyr Thr 20 25 30 Asn Gly Glu Glu Thr Val Arg Phe Asp Ser Asp TrpGly Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Gln Asp Ala Glu TyrTrp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Glu Lys Arg Ala Glu Val AspArg Val Cys Arg 65 70 75 80 His Asn Tyr Gly Gly Met Glu Ser Phe Thr ValGln Arg Arg 85 90 60 94 PRT BOVINE 60 Arg Glu Ile Gln Pro His Phe LeuGlu Tyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu ArgVal Arg Phe Leu Asn Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val ArgPhe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgAla Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg AlaArg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val GlyGlu Ser Phe Thr Val Gln Arg Arg 85 90 61 94 PRT BOVINE 61 Arg Glu IleGln Pro His Phe Leu Gln Tyr His Lys Gly Glu Cys His 1 5 10 15 Phe PheAsn Gly Thr Glu Arg Val Arg Leu Leu Asp Arg His Phe Tyr 20 25 30 Asn GlyGlu Glu Tyr Val Arg Phe Asp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala ValThr Glu Leu Gly Arg Pro Ser Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys AspPhe Leu Glu Arg Arg Arg Ala Glu Val Asp Thr Val Cys Arg 65 70 75 80 HisAsn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 62 94 PRTBOVINE 62 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg TyrPhe His 20 25 30 Asn Gly Glu Glu Phe Leu Arg Phe Asp Ser Asp Trp Gly GluTyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp AsnSer Gln 50 55 60 Lys Glu Ile Leu Glu Arg Ala Arg Ala Ala Val Asp Thr TyrCys Arg 65 70 75 80 His Asn Tyr Gly Gly Val Glu Ser Phe Thr Val Gln ArgArg 85 90 63 94 PRT BOVINE 63 Arg Glu Ile Gln Pro His Phe Leu Glu TyrSer Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val ArgPhe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly Glu Glu Thr Val Arg Phe AspSer Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Gln AspAla Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Glu Lys Arg AlaGlu Val Asp Arg Val Cys Arg 65 70 75 80 His Asn Tyr Gly Gly Met Glu SerPhe Thr Val Gln Arg Arg 85 90 64 94 PRT BOVINE 64 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Asn Arg Tyr Phe His 20 25 30 Asn Gly Glu GluPhe Val Arg Phe Gly Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr GluLeu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile LeuGlu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn TyrGly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 65 94 PRT BOVINE 65Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 1015 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe His 20 2530 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 4045 Ala Val Thr Glu Leu Gly Gln Arg Val Ala Glu Tyr Cys Asn Ser Gln 50 5560 Lys Asp Phe Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 7075 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 6694 PRT BOVINE 66 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser GluCys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Glu ArgSer Phe Tyr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp GlyGlu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr TrpAsn Ser Gln 50 55 60 Lys Asp Asp Leu Glu Glu Arg Arg Ala Glu Val Asp ArgVal Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val GlnArg Arg 85 90 67 94 PRT BOVINE 67 Arg Glu Ile Gln Pro His Phe Leu GluTyr Ser Thr Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Phe Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg PheAsp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg ProAsp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg Ala ArgAla Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Val GluSer Phe Thr Val Gln Arg Arg 85 90 68 94 PRT BOVINE 68 Arg Glu Ile GlnPro His Phe Leu Glu Tyr Cys Lys Arg Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly GluGlu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Pro Ala Ala Lys Gln Trp Asn Ser Gln 50 55 60 Lys Asp PheLeu Glu Glu Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His AsnTyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 69 94 PRT BOVINE69 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 510 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Glu Arg Ser Phe Tyr 2025 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 3540 45 Ala Val Thr Glu Leu Gly Arg Arg Val Ala Glu Gln Leu Asn Ser Gln 5055 60 Lys Asp Phe Leu Glu Gln Lys Arg Ala Asn Val Asp Thr Tyr Cys Arg 6570 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 9070 94 PRT BOVINE 70 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Thr Lys LysGlu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu AspArg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp TrpGly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Lys TyrArg Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Glu Lys Arg Ala Gln Val AspThr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr ValGln Arg Arg 85 90 71 94 PRT BOVINE 71 Arg Glu Ile Gln Pro His Phe LeuGln Tyr His Lys Gly Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu ArgVal Arg Leu Leu Asp Arg His Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val ArgPhe Asp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgPro Ser Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg ArgArg Ala Glu Val Asp Thr Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val ValGlu Ser Phe Thr Val Gln Arg Arg 85 90 72 94 PRT BOVINE 72 Arg Glu IleGln Pro His Phe Leu Glu Tyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe PheAsn Gly Thr Glu Arg Val Arg Phe Leu Asn Arg Tyr Phe His 20 25 30 Asn GlyGlu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala ValThr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys AspIle Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 HisAsn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 73 94 PRTBOVINE 73 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg TyrPhe His 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp Trp Gly GluTyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Gln Arg Val Ala Glu Tyr Cys AsnSer Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala Arg Ala Ala Val Asp Thr TyrCys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln ArgArg 85 90 74 94 PRT BOVINE 74 Arg Glu Ile Gln Pro His Phe Leu Glu TyrSer Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val ArgPhe Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe AspSer Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Gln Arg ValAla Glu Tyr Ser Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala Arg AlaAla Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu SerPhe Thr Val Gln Arg Arg 85 90 75 94 PRT BOVINE 75 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu GluTyr Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr GluLeu Gly Gln Arg Val Ala Glu Tyr Cys Asn Ser Gln 50 55 60 Lys Asp Phe LeuGlu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn TyrGly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 76 94 PRT BOVINE 76Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 1015 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe His 20 2530 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 4045 Ala Val Thr Glu Leu Gly Gln Arg Val Ala Glu Tyr Cys Asn Ser Gln 50 5560 Lys Asp Phe Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 7075 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 7794 PRT BOVINE 77 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser GluCys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp ArgTyr Tyr Thr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp GlyGlu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Gln TrpAsn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Ser Arg Arg Thr Ala Val Asp ThrTyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Phe Glu Ser Phe Thr Val GlnArg Arg 85 90 78 94 PRT BOVINE 78 Arg Glu Ile Gln Pro His Phe Leu GluTyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Phe Leu Glu Arg Ser Phe Tyr 20 25 30 Asn Gly Glu Glu Asn Val Arg PheAsp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg ProAsp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Glu Arg ArgAla Glu Val Asp Arg Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly GluSer Phe Thr Val Gln Arg Arg 85 90 79 94 PRT BOVINE 79 Arg Glu Ile GlnPro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Tyr Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly GluGlu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Arg Val Ala Glu Gln Leu Asn Gly Gln 50 55 60 Lys Asp ThrLeu Glu Arg Glu Arg Ala Tyr Val Asp Tyr Tyr Cys Arg 65 70 75 80 His AsnTyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 80 94 PRT BOVINE80 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 510 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Tyr Leu Asp Arg Tyr Phe His 2025 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 3540 45 Ala Val Thr Glu Leu Gly Arg Arg Val Ala Glu Gln Leu Asn Gly Gln 5055 60 Lys Asp Thr Leu Glu Arg Glu Arg Ala Tyr Val Asp Tyr Tyr Cys Arg 6570 75 80 His Asn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 9081 94 PRT BOVINE 81 Arg Glu Ile Gln Pro His Phe Leu Gln Tyr His Lys GlyGlu Cys His 1 5 10 15 Phe Leu Asn Gly Thr Glu Arg Val Arg Leu Leu AspArg His Phe His 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp TrpAsp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Ser Ala Glu TyrTrp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Arg Arg Ala Glu Val AspThr Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val Val Glu Ser Phe Thr ValGln Arg Arg 85 90 82 94 PRT BOVINE 82 Arg Glu Ile Gln Pro His Phe LeuGlu Tyr His Lys Gly Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu ArgVal Arg Leu Leu Asp Arg Tyr Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val ArgPhe Asp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgPro Ser Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg ArgArg Ala Glu Val Asp Thr Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val GlyGlu Ser Phe Thr Val Gln Arg Arg 85 90 83 94 PRT BOVINE 83 Arg Glu IleGln Pro His Phe Leu Glu Tyr His Lys Ser Glu Cys Arg 1 5 10 15 Phe PheHis Gly Thr Glu Arg Val Arg Tyr Leu Asp Arg Tyr Phe Tyr 20 25 30 Asn GlyGlu Glu Tyr Val Arg Phe Asp Asn Asp Trp Gly Glu Phe Arg 35 40 45 Ala ValAla Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys AspPhe Leu Glu Arg Lys Arg Ala Glu Val Asp Thr Tyr Cys Arg 65 70 75 80 HisAsn Tyr Gly Val Ile Glu Ser Phe Thr Val Gln Arg Arg 85 90 84 94 PRTBOVINE 84 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg TyrTyr Thr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp Gly GluPhe Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Gln Trp AsnSer Gln 50 55 60 Lys Asp Phe Leu Glu Ser Arg Arg Thr Ala Val Asp Thr TyrCys Arg 65 70 75 80 His Asn Tyr Gly Val Phe Glu Ser Phe Thr Val Gln ArgArg 85 90 85 94 PRT BOVINE 85 Arg Glu Ile Gln Pro His Phe Leu Glu TyrSer Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val ArgPhe Leu Asp Arg Tyr Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe AspSer Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Ala Glu Leu Gly Arg Pro GluAla Lys Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Ile Leu Glu Arg Lys Arg AlaAla Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Ile Glu SerPhe Thr Val Gln Arg Arg 85 90 86 94 PRT BOVINE 86 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Tyr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly Glu GluThr Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Leu Thr GluLeu Gly Arg Gln Asp Ala Glu Gln Trp Asn Ser Gln 50 55 60 Lys Asp Ile LeuGlu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 Tyr Asn TyrGly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 87 94 PRT BOVINE 87Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Arg Glu Cys His 1 5 1015 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe His 20 2530 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 4045 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp His Ser Gln 50 5560 Lys Glu Ile Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 7075 80 His Asn Tyr Gly Gly Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 8894 PRT BOVINE 88 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Tyr Lys Ser GluCys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp ArgTyr Tyr Thr 20 25 30 Asn Gly Gly Glu Thr Val Arg Phe Asp Ser Asp Trp GlyGlu Phe Arg 35 40 45 Ala Leu Thr Glu Leu Gly Arg Gln Asp Ala Gly Gln TrpAsn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala Arg Ala Ala Val Asp ThrTyr Cys Arg 65 70 75 80 Tyr Asn Tyr Gly Val Gly Glu Ser Phe Thr Val GlnArg Arg 85 90 89 94 PRT BOVINE 89 Arg Glu Ile Gln Pro His Phe Leu GluTyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Leu Leu Glu Arg Tyr Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val Arg PheAsp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Ala Glu Leu Gly Arg ProAsp Ala Lys Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Ser Arg ArgThr Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly GluSer Phe Thr Val Gln Arg Arg 85 90 90 94 PRT BOVINE 90 Arg Glu Ile GlnPro His Phe Leu Glu Tyr Thr Lys Ser Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly GluGlu Asn Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Pro Asp Ala Glu Gln Trp Asn Ser Gln 50 55 60 Lys Asp PheLeu Glu Ser Arg Arg Thr Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His AsnTyr Gly Val Phe Glu Ser Phe Thr Val Gln Arg Arg 85 90 91 94 PRT BOVINE91 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 510 15 Phe Ser Asn Gly Thr Glu Arg Val Arg Leu Leu Asp Arg Tyr Phe His 2025 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 3540 45 Ala Val Ala Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 5055 60 Lys Asp Phe Leu Glu Ser Arg Arg Thr Ala Val Asp Thr Tyr Cys Arg 6570 75 80 His Asn Tyr Gly Val Ile Glu Ser Phe Thr Val Gln Arg Arg 85 9092 94 PRT BOVINE 92 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr SerGlu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Tyr Leu AspArg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp TrpGly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Arg Val Ala Glu GlnLeu Asn Gly Gln 50 55 60 Lys Asp Thr Leu Glu Arg Glu Arg Ala Tyr Val AspThr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Val Glu Ser Phe Thr ValGln Arg Arg 85 90 93 94 PRT BOVINE 93 Arg Glu Ile Gln Pro His Phe LeuGlu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu ArgVal Arg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly Glu Glu Asn Val ArgPhe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgPro Glu Ala Lys Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile Leu Glu Arg LysArg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val IleGlu Ser Phe Thr Val Gln Arg Arg 85 90 94 94 PRT BOVINE 94 Arg Glu IleGln Pro His Phe Leu Glu Tyr Tyr Lys Ser Glu Cys His 1 5 10 15 Phe PheAsn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn GlyGlu Glu Thr Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala LeuThr Glu Leu Gly Arg Gln Asp Ala Glu Gln Trp Asn Ser Gln 50 55 60 Lys AspPhe Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 HisAsn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 95 94 PRTBOVINE 95 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Tyr Lys Ser Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg TyrTyr Thr 20 25 30 Asn Gly Glu Glu Thr Val Arg Phe Asp Ser Asp Trp Gly GluPhe Arg 35 40 45 Ala Leu Thr Glu Leu Gly Arg Gln Asp Ala Glu Gln Trp AsnSer Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala Arg Ala Ala Val Asp Thr TyrCys Arg 65 70 75 80 Tyr Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln ArgArg 85 90 96 94 PRT BOVINE 96 Arg Glu Ile Gln Pro His Phe Leu Glu TyrSer Thr Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val ArgTyr Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe Val Arg Phe AspSer Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Arg ValAla Glu Gln Leu Asn Gly Gln 50 55 60 Lys Asp Thr Leu Glu Arg Glu Arg AlaTyr Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Val Glu SerPhe Thr Val Gln Arg Arg 85 90 97 94 PRT BOVINE 97 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly Glu GluThr Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Thr GluLeu Gly Arg Gln Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe LeuGlu Glu Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn TyrGly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 98 94 PRT BOVINE 98Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 5 1015 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Tyr Thr 20 2530 Asn Gly Glu Glu Thr Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 4045 Ala Val Thr Glu Leu Gly Arg Gln Asp Ala Glu Tyr Trp Asn Ser Gln 50 5560 Lys Asp Phe Leu Glu Glu Lys Arg Ala Glu Val Asp Arg Val Cys Arg 65 7075 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90 9994 PRT BOVINE 99 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys Ser GluCys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Glu ArgSer Phe Tyr 20 25 30 Asn Gly Glu Glu Asn Val Arg Phe Asp Ser Asp Trp GlyGlu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Arg Arg Val Ala Glu Gln LeuAsn Ser Gln 50 55 60 Lys Asp Leu Leu Glu Ala Lys Arg Ala Asn Val Asp ThrTyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val GlnArg Arg 85 90 100 94 PRT BOVINE 100 Arg Glu Ile Gln Pro His Phe Leu GluTyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg ValArg Leu Leu Glu Arg Tyr Phe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val Arg PheAsp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val Ala Glu Leu Gly Arg ProAsp Ala Lys Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Ser Arg ArgThr Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly GluSer Phe Thr Val Gln Arg Arg 85 90 101 94 PRT BOVINE 101 Arg Glu Ile GlnPro His Phe Leu Glu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe AsnGly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly GluGlu Asn Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 40 45 Ala Val ThrGlu Leu Gly Arg Pro Asp Ala Glu Gln Trp Asn Ser Gln 50 55 60 Lys Asp PheLeu Glu Ser Arg Arg Thr Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His AsnTyr Gly Val Phe Glu Ser Phe Thr Val Gln Arg Arg 85 90 102 94 PRT BOVINE102 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Thr Lys Lys Glu Cys His 1 510 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asn Arg Tyr Phe His 2025 30 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 3540 45 Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 5055 60 Lys Glu Ile Leu Glu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 6570 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg 85 90103 94 PRT BOVINE 103 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser LysSer Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe LeuAsp Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser AspTrp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Gln Arg Asp Ala GluTyr Cys Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala Arg Ala Ala ValAsp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe ThrVal Gln Arg Arg 85 90 104 94 PRT BOVINE 104 Arg Glu Ile Gln Pro His PheLeu Glu Tyr Thr Lys Lys Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr GluArg Val Arg Phe Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Phe ValArg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu GlyArg Pro Asp Ala Lys Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu ArgAla Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly ValGly Glu Ser Phe Thr Val Gln Arg Arg 85 90 105 94 PRT BOVINE 105 Arg GluIle Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 5 10 15 PhePhe Asn Gly Thr Glu Arg Val Arg Tyr Leu Asp Arg Tyr Phe His 20 25 30 AsnGly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 AlaVal Thr Glu Leu Gly Arg Arg Val Ala Glu Gln Leu Asn Gly Gln 50 55 60 LysAsp Thr Leu Glu Arg Glu Arg Ala Tyr Val Asp Thr Tyr Cys Arg 65 70 75 80His Asn Tyr Gly Val Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 106 94 PRTBOVINE 106 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr His Lys Ser Glu CysHis 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg TyrPhe Tyr 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp Trp Gly GluPhe Arg 35 40 45 Ala Val Ala Glu Leu Gly Arg Pro Glu Ala Lys Tyr Trp AsnSer Gln 50 55 60 Lys Glu Ile Leu Glu Arg Lys Arg Ala Ala Val Asp Thr TyrCys Arg 65 70 75 80 His Asn Tyr Gly Val Ile Glu Ser Phe Thr Val Gln ArgArg 85 90 107 94 PRT BOVINE 107 Arg Glu Ile Gln Pro His Phe Leu Glu TyrTyr Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val ArgPhe Leu Asp Arg Tyr Tyr Thr 20 25 30 Asn Gly Glu Glu Thr Val Arg Phe AspSer Asp Trp Gly Glu Phe Arg 35 40 45 Ala Leu Thr Glu Leu Gly Arg Gln AspAla Glu Gln Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala Arg AlaAla Val Asp Thr Tyr Cys Arg 65 70 75 80 Tyr Asn Tyr Gly Val Gly Glu SerPhe Thr Val Gln Arg Arg 85 90 108 94 PRT BOVINE 108 Arg Glu Ile Gln ProHis Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 5 10 15 Phe Phe Asn GlyThr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu GluPhe Val Arg Phe Asp Ser Asp Trp Gly Glu Tyr Arg 35 40 45 Ala Val Thr GluLeu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Glu Ile LeuGlu Arg Ala Arg Ala Ala Val Asp Thr Tyr Cys Arg 65 70 75 80 His Asn TyrGly Gly Val Glu Ser Phe Thr Val Gln Arg Arg 85 90 109 94 PRT BOVINE 109Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Thr Ser Glu Cys His 1 5 1015 Phe Ser Asn Gly Thr Glu Arg Val Arg Leu Leu Asp Arg Tyr Phe His 20 2530 Asn Gly Glu Glu Phe Val Arg Phe Asp Ser Asp Trp Gly Glu Phe Arg 35 4045 Ala Val Ala Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln 50 5560 Lys Asp Phe Leu Glu Ser Arg Arg Thr Ala Val Asp Thr Tyr Cys Arg 65 7075 80 His Asn Tyr Gly Val Ile Glu Ser Phe Thr Val Gln Arg Arg 85 90 11094 PRT BOVINE 110 Arg Glu Ile Gln Pro His Phe Leu Glu Tyr Ser Lys SerGlu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu AspArg Tyr Phe His 20 25 30 Asn Gly Glu Glu Tyr Val Arg Phe Asp Ser Asp TrpGly Glu Tyr Arg 35 40 45 Ala Val Thr Glu Leu Gly Gln Arg Val Ala Glu TyrCys Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg Ala Arg Ala Ala Val AspThr Tyr Cys Arg 65 70 75 80 His Asn Tyr Gly Val Gly Glu Ser Phe Thr ValGln Arg Arg 85 90 111 94 PRT BOVINE 111 Arg Glu Ile Gln Pro His Phe LeuGlu Tyr Ser Lys Ser Glu Cys His 1 5 10 15 Phe Phe Asn Gly Thr Glu ArgVal Arg Phe Leu Asp Arg Tyr Phe His 20 25 30 Asn Gly Glu Glu Tyr Val ArgPhe Asp Ser Asp Trp Asp Glu Phe Arg 35 40 45 Ala Val Thr Glu Leu Gly ArgPro Ser Ala Glu Tyr Trp Asn Ser Gln 50 55 60 Lys Asp Phe Leu Glu Arg ArgArg Ala Glu Val Asp Thr Val Cys Arg 65 70 75 80 His Asn Tyr Gly Val ValGlu Ser Phe Thr Val Gln Arg Arg 85 90 112 4 PRT BOVINE 112 Val Asp ThrVal 1 113 4 PRT BOVINE 113 Val Asp Arg Val 1 114 18 DNA ArtificialSequence Description of Artificial Sequence PRIMER 114 tgtaaaacgacggccagt 18 115 18 DNA Artificial Sequence Description of ArtificialSequence PRIMER 115 caggaaacag ctatgacc 18

What is claimed is:
 1. A method for judging a possibility of onset of bovine leukemia caused by bovine leukemia virus BLV, wherein a bovine individual, in which an amino acid sequence defined by amino acid numbers 75 to 78 of β1 domain of bovine MHC Class II DRβ chain is Val-Asp-Thr-Tyr, is judged to have a possibility of the onset of the bovine leukemia.
 2. The method according to claim 1 wherein a bovine individual, in which an amino acid sequence defined by amino acid numbers 75-78 of β1 domain of bovine MHC Class II DRβ chain is Val-Asp-Thr-Tyr in both alleles, is judged to have a risk of the onset.
 3. A method for judging a possibility of onset of bovine leukemia caused by bovine leukemia virus BLV, which comprises: (1) amplifying genome DNA isolated from a bovine individual by polymerase chain reaction (PCR) to prepare a PCR product containing a DNA coding for a part or full length of β1 domain of bovine MHC Class II DRβ chain, and (2) judging that the bovine individual, in which an amino acid sequence corresponding to amino acid numbers 75 to 78 of the β1 domain of the bovine MHC Class II DRβ chain is Val-Asp-Thr-Tyr in an amino acid sequence encoded by the DNA contained in the PCR product, has a possibility of onset of the bovine leukemia.
 4. The method according to claim 3 which comprises digesting the PCR product by using PstI.
 5. The method according to claim 3 wherein a bovine individual, in which an amino acid sequence defined by amino acid numbers 75 to 78 of β1 domain of bovine MHC Class II DRβ chain is Val-Asp-Thr-Tyr in both alleles, is judged to have a risk of the onset. 